The P-Glycoprotein multidrug transport protein is a membrane glycoprotein which is overexpressed in cell lines and tumors that are resistent to structurally unrelated cytotoxic agents. The exact mechanism of action is unknown; however, the overexpression of the protein results in reduced uptake of cytotoxic drugs. We have been investigating the interaction of forskolin, a naturally occurring diterpene, with the P-Glycoprotein. Forskolin photoaffinity agents have been synthesized which are capable of covalent labelling of the forskolin binding site. The location of the forskolin labelling sites on the P-Glycoprotein is being determined by digestion of labelled protein and immunoprecipitation with peptide-specific antibodies. Labelling sites have been identified on the C- and N-terminal halves of the protein. The labelling site on the N-terminal half of the protein has been associated with a 68 amino acid fragment that spans the fifth and sixth transmembrane regions of the P-Glycoprotein. The labelling patterns identified with forskolin photoaffinity labels are identical to those observed with photoaffinity labels derived from another drug, prazosin. The specific labelling site on the 68 amino acid fragment will be determined by amino acid sequencing of the isolated fragment.